Breast cancer is the most common form of cancer occurring in females in the United States. The incidence of breast cancers in the United States is projected to be 180,200 cases diagnosed and 43,900 breast cancer related deaths to occur during 1997 (American Cancer Society statistics). The incidence of breast cancers in the United States is projected to be 213,910 cases diagnosed and 40,921 breast cancer related deaths to occur during 2004 (American Cancer Society statistics). Worldwide, the incidence of breast cancer has increased from 700,000 in 1985 to about 1,050,000 in 2000. G. N. Hortobagyi et al., C A Cancer J. Clin. 45: 199-226 (1995), Parkin D M., Lancet Oncol. 2(10):596 (2001).
Procedures used for detecting, diagnosing, staging, monitoring, prognosticating, preventing, treating or determining predisposition to diseases or conditions of the breast, such as breast cancer, are of critical importance to the outcome of the patient. For example, patients diagnosed with early breast cancer have greater than a 90% five-year relative survival rate as compared to a survival rate of about 20% of patients diagnosed with distantly metastasized breast cancers (American Cancer Society statistics). Currently, the best initial indicators of early breast cancer are physical examination of the breast and mammography. J. R. Harries et al. In: Cancer: Principles and Practice of Oncology, Fourth Edition, pp. 1264-1332, Philadelphia, Pa.: J/B. Lippincott Co. (1993). Mammography may detect a breast tumor before it can be detected by physical examination, but it has limitations. For example, mammography's predictive value depends on the observer's skill and the quality of the mammogram. In addition, 80% to 93% of suspicious mammograms are false positives, and 10% to 15% of women with breast cancer have false negative mammograms. C. J. Wright et al., Lancet 346: 29-32 (1995). Thereupon, new diagnostic methods which are more sensitive and specific for detecting early breast cancer are clearly needed.
A major concern with any cancer, including breast cancer, is the spread of the disease from a localized area (such as the breast) to other parts of the body (known as metastasis). Metastasis is believed to have occurred when epithelial cells are detected in the hematopoietic system. This discovery is important since metastasis is diagnostic of certain stages of cancer, and decisions concerning the proper treatment of a cancer patient are largely dependent upon properly characterizing the stage of the disease. In particular, the treatment of patients having localized cancer can be vastly different from the treatment of patients in metastatic stages of cancer.
Early efforts to detect the spread of cancer by detecting epithelial cells in the hematopoietic system included immunocytological assay procedures. Unfortunately, these methods are largely inaccurate because antibodies used in these assays that are ostensibly specific for epithelial cells, demonstrate cross-reactivity for cells normally found in the hematopoietic system. Hence, “normal hematopoietic cells” are sometimes detected in the absence of metastatic cells and therefore, false positive results can be obtained according to these assay procedures. Additionally, immunocytological assays lack sensitivity and can produce false negative results when low levels of epithelial cells are actually present in the hematopoietic system. Accordingly, early stages of metastatic cancer can be misdiagnosed using immunocytological assays.
With the advent of nucleic acid amplification reactions such as the polymerase chain reaction (PCR), epithelial cells present in the hematopoietic system can be detected via the nucleic acid instead of the protein. Hence, problems associated with cross-reactive antibodies are avoided. Additionally, it is well known that nucleic acid amplification reactions are significantly more sensitive than more conventional antibody based assay methods. Amplification based assays for detecting epithelial cells in the blood stream have therefore provided significant advantages over immunocytological assay methods for detecting early stages of metastatic cancer.
PCR based assays employed to detect epithelial cells in the hematopoietic system have been reported in the literature. Many of these assays target a nucleic acid sequence encoding cytokeratin 19 (CK19), a protein found on the surface of epithelial cells. However, pseudogenes (comprising a nucleic acid sequence that closely mimics the gene for CK19) are present in the human genome. Thus, one challenge facing those developing amplification assays to detect a CK19 target sequence is to design assays that amplify and detect a sequence from the CK19 gene but not the closely related pseudogene.
In addition to CK19, two other markers have been discovered that may be used to detect metastatic breast cancer cells. These two markers are termed BU101 (also called lipophilin B) and BS106 (also called small breast epithelial mucin or “SBEM”). (Colpitts et al., Ann. NY Acad. Sci. 923:312-315 (2000); Colpitts et al., Tumor Biol. 23:263-278 (2002)). These markers were found to be specifically expressed in breast epithelium. Measurements of the expression of these two markers have been made and compared to CK19. CK19 has been found to be highly sensitive in detecting all breast cancers, while BU101 and BS106 were found to be slightly more restrictive. The reason for this is that CK19 is highly expressed by most epithelial cells making it a very sensitive marker, but it cannot be considered breast specific. Thereupon, BU101 and BS106 are considered to be more breast specific markers compared to CK19. Therefore, BU101 and BS106 may provide sufficient discrimination to detect and measure occult breast cancer.
The cDNA sequence of BS106 has been studied and characterized. (Colpitts et al., Tumor Biol. 23:263-278 (2002)). It has been demonstrated that BS106 is expressed in mammary, salivary and prostate glands, but not in other tissues. The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein, based on amino acid composition, extensive O-linked glycosylation, and expression profile. BS106 mRNA has been detected in 90% of the breast tissues examined.
BS106 mRNA expression has been detected in more than 90% of invasive ductal carcinomas. It was found that BS106 mRNA is expressed in breast cancer cell lines but not in cell lines of non-breast origin. This indicates that BS106 expression is a common feature of breast cancer and can serve as a useful marker for breast nodal metastasis, both for detection of micrometastatic cells within lymph nodes as well as in differential diagnosis of the primary origin of unknown metastasis.
However, known BS106 marker detection methods are cumbersome and non-specific. Some methods use a two-step cDNA production and then PCR while some methods use gel detection (TaqMan® PCR).
Additionally, another problem with known BS106 marker detection methods is that the primers and probes employed in such methods are not very specific or sensitive. In fact, it is well known that amplification primer sequences that are used for such detection methods can be selected based upon computer comparisons of closely related sequences. Theoretically, sequences selected in this manner effectively should produce copies of the selected target sequence when employed according to nucleic acid amplification principles. Notwithstanding the theoretical efficacy of sequences selected in the above manner, it is often times true that such sequences do not produce acceptable amounts of amplification product. Unfortunately, this phenomenon is not well understood. Accordingly, while primers initially can be screened using computer programs, efficacy cannot be adequately determined until such primers are employed in practice. This was especially true in the design of primers and probes for use in the amplification of BS106 mRNA. The inventors of the present invention found the design of primers and probes for use in the amplification of BS106 mRNA to be difficult because BS106 mRNA contains rather small introns, repetitive sequence strings and AT regions. Many of the primer pairs and probes selected by a computer program (such as OLIGO™) could not be used because these selections did not span the introns and had a high false priming potential with other human genes.
Therefore, there is a need in the art for a detection method that is simple and specific for a BS106 marker. Additionally, in connection with said detection method, there is a need for primer and probe sequences that can be used in said method and are highly specific and sensitive for the BS106 marker.